Hormone-secreting cells are highly differentiated and specialized for the synthesis and secretion of typically one or two specific hormones. Examples of hormone-secreting cells include certain cells of the pituitary gland, the endometrium, the ovary and the pancreas. The pituitary gland contains cells specialized for the synthesis and secretion of glycoprotein hormones known as gonadotrophins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which act on the gonads. The gonadotrophins secreted by the pituitary enter the blood stream and reach the gonads, where they exert their affects. Within the ovary, upon stimulation with gonadotrophins, granulosa cells surrounding an ovum differentiate within the preovulatory follicle to synthesize and secrete estrogen and progesterone. Specialized cells of the endometrium also synthesize and secrete estrogen and progesterone. Within the pancreas, .beta.-cells of the islets respond to increased blood glucose concentration with an increase in insulin secretion.
Conventional cell culture technology is sufficient for the propagation of certain cell types in vitro such as fibroblasts taken from normal tissue or from tumors. It has long been a goal of scientists to maintain hormone-secreting cells in vitro, however standard culture conditions do not promote the long-term survival or proliferation of hormone-secreting cells. For practical purposes, it would be desirable to establish in culture cells which both proliferate and perform their specialized functions, i.e., synthesis and secretion of specific hormones.
For primary tissue culture, normal or tumor cells are removed from an animal or a human cell donor, placed in a liquid chemical medium in laboratory culture dishes, and maintained in an incubator under physical conditions which mimic the cells' environment in vivo. The medium and the incubator environment provide regulated temperature, pH, nutrients, growth factors, protection against pathogens, and in some cases a necessary substrate for cell attachment. Even under optimized culture conditions, however, most types of normal cells have a limited life span in culture. Typically, when cells other than fibroblasts are established in primary tissue culture they do not proliferate; they may or may not continue to perform their differentiated functions over the short-term. When the cells reach the end of their natural life-span they die, thus the cultures are self-limiting. Hormone-secreting cells generally survive in culture for no more than 8 to 12 days, during which time they undergo few or no cycles of cell division. During the life-span of hormone-secreting cells in culture, as they have been maintained using prior known techniques, such cells typically undergo a loss of function as evidenced by a decrease in hormone production.
In order to increase the life-span of hormone-secreting cells in culture, published techniques have included the use of embryonic cells. The strategy of starting with embryonic cells is based on the fact that embryonic cells are relatively less differentiated than adult cells, and thus can be expected to go through several cycles of cell division before becoming terminally differentiated, i.e., specialized for hormone synthesis. It is an axiom of biology that undifferentiated cells proliferate at a greater rate than differentiated cells. It is generally believed that by the time a cell has developed the necessary intra-cellular machinery for hormone synthesis and secretion, it is no longer able to divide rapidly, if at all.
Another known strategy for establishing cells in culture is to start with cancer cells, since cancer cells would be expected to have a greater potential for proliferation. However, few cells derived from tumors or other cancerous lesions are able to become established and divide in culture. One cell line was established from a malignant human choriocarcinoma by propagating the tumor cells through 304 serial transplantations to the hamster cheek pouch over a period of 8 years before establishment in vitro (BeWo cell line; ATCC CCL 98; May 1990 supplement to the 1988 American Tissue Culture Collection ATCC! catalog of cell lines). The BeWo cell line was reported to produce human chorionic gonadotrophin (hCG), polypeptide hormones, human placental lactogen (hPL), estrogens and progestins. A cell line with an abnormal karyotype was established from the malignant ascites of a patient with adenocarcinoma of the ovary (NIH:OVCAR-3; ATCC HTB 161; ref. supra). The OVCAR-3 cell line was reported to possess androgen and estrogen receptors, however no synthesis of hormones by these cells was reported.
A rat clonal beta-cell line (RIN) was established in culture from a rat insulinoma (Clark, S. A., et al, 1980, Endocrinology 127: 2779-2788). RIN cells were reported to secrete insulin in vitro in response to low levels of glucose, with maximal response at 0.6 mM glucose. This response is comparable to that of immature rat beta-cells, and quite different from that of normal mature rat islets which secrete in response to glucose concentrations ranging from 5 mM to 16 mM.
It is apparent from the forgoing that tumor cells are difficult to establish in vitro. Moreover, tumor cells that do become established in culture often possess abnormal characteristics which diminish their usefulness, such as the loss or alteration of hormone synthesis or secretogogue responsiveness.
Using a strategy based on the notion that abnormal cells are more likely to grow in vitro, normal cells have been transformed in culture by various means including the use of UV light, chemical carcinogens, and the introduction of oncogenes. Rat granulosa cells were transformed by co-transfection with the entire SV 40 genome and the activated Ha-ras gene (Baum, G., et al. 1990 Develop Biol 112: 115-128). These cells were reported to retain at least some differentiated characteristics, i.e., they were able to synthesize steroids in response to cAMP.
Other cell lines established in culture include UMR cells, derived from normal islets of neonatal rats (NG, K. W., et al., 1987, J. Endocrinol. 113: 8-10) and HIT cells, derived by simian virus-40 infection of hamster islets (Santerre, R. F., et al., 1981, PNAS 78: 4339-4343). The insulin secretory output of these cell lines is low, and response to glucose is lost with passage in culture.
In order to promote the selection of non-transformed hormone-secreting cells as starting material for culture, a regimen of hormone treatment in vivo was used before removal of cells from the donor (Amsterdam, A., et al. 1989 Endocrinology 124: 1956-1964). Cells were obtained from ovarian follicles removed from women who had received hormonal therapy in preparation for in vitro fertilization. For additional promotion of differentiated function, cells were maintained on extra-cellular matrix and further treated with human chorionic gonadotrophin (hCG). Although the cells had a differentiated appearance and secreted progesterone in culture, the cells were reported to survive in culture for only five days. In a similar study, cells were reported to survive for eight days (Pellicer, A., et al. 1990 Fertility and Sterility 54: 590-596).
Another strategy for promoting the maintenance of differentiation in culture involved the culturing of the component parts of entire follicles, including the oocyte and cumulus complex (Vanderhyden, B. C., et al. 1990 Develop. Biol. 140: 307-317). In this type of "combination culture", mouse granulosa cells were maintained in a differentiated state for 7 days.
The above description of the state-of-the-art makes it apparent that there is a need for methods to maintain and propagate hormone-secreting cells in long-term cultures. Such cultures could be developed as biological "factories" for the production of therapeutically useful hormones. Well-established hormone-secreting cell lines would also offer the possibility of in vitro bio-assays based on the cells' responses to drugs such as gonadotrophin preparations. In addition, such cell lines would offer the possibility of in vitro bio-assays for the toxicity of drugs and other chemicals. Established cell lines would also be candidates for implantation to correct diseases due to hormone deficiencies. For instance, diabetics could be stabilized and possibly cured through the implantation of cells which replace the function of insulin-secreting beta-cells of the pancreas.
There exists a need for methods to produce consistent physiologically correct preparations of gonadotrophin hormones. Human gonadotrophin preparations (hMG), which typically contain both FSH and LH, are administered to women who are undergoing pre-treatment leading to in vitro fertilization. The administered hMG stimulates the woman's ovaries to produce multiple pre-ovulatory follicles, which are subsequently aspirated for in vitro fertilization. hMG is currently derived from the urine of post-menopausal women. Each lot differs according to the age and endocrine status of the urine donors, the differences being in both concentration and types of isoforms present in the final product. There are at least 11 isoforms of human follicle-stimulating hormone (hFSH) and 7 isoforms of human luteinizing hormone (hLH) (Stone, B. A., et al. 1990 Acta Endo (Copenhagen) 123: 161-168). Analysis by high-performance liquid chromatography (HPLC) of various hMG preparations showed between-lot variability in the presence and concentration of isoforms of FSH (Stone, B. A. et al, supra). Different isoforms have different biopotencies (Gharib, S. D., et al. 1990 In: Endocrine Reviews 11: 177-199). Since certain isoforms of FSH are more biopotent than others, there is between-lot variability in biopotency among various hMG preparations. Moreover, the presence of LH isoforms in a preparation affects the biopotency of FSH present in the preparation.
Scientists are currently attempting to produce genetically engineered FSH of a desired and consistent biopotency. There is a clear need for a cost-effective assay to enable the development of therapeutically useful preparations of genetically engineered gonadotrophins.
There exist two major forms of chemical assay for gonadotrophins: HPLC and radioimmunoassay (RIA). The HPLC technique is precise but does not identify which chemical properties of hMG preparations relate to biopotency. Moreover, the HPLC technique requires considerable technical expertise, instrumentation, and investment of technical labor. Tests based on immunologic recognition of a gonadotrophin (RIA) are limited by the inherent cross-reactivity of the antibodies with disparate isoforms of the gonadotrophins. For instance, a single RIA numerical value for FSH concentration could include several FSH isoforms of differing biopotency. Thus the current techniques for chemical assay do not provide a means to assess the biopotency of a therapeutic preparation of gonadotrophin.
The need for biopotency assessments of gonadotrophins has been acknowledged by several national agencies, including the U.S. Food and Drug Administration (FDA). The assays currently accepted by the FDA are in vivo assays conducted in rodents. The in vivo assay for FSH is the Steelman-Pohley assay which is based on mouse uterine weight gain. One in vivo assay for LH is the rat Leydig cell assay; the degree of proliferation in the seminal vesicles of the immature male rat is the index for assessing biopotency of LH. Another in vivo bioassay for LH is the rat ovarian ascorbic acid depletion test. These in vivo assays are disadvantageous because they require the sacrifice of large numbers of laboratory animals. For instance, the sacrifice of 2,000 mice is required to measure the stability factor for one particular batch of hMG. This figure of 2,000 mice does not include the number required to establish the biopotency of the original batch. The need for a more cost-effective bioassay is apparent. Moreover, the results from tests conducted on rodent cells are not necessarily applicable to biopotency in humans.
The current source for therapeutic gonadotrophins, while convenient, is limited by the inherent biological variability among the human donors. The major source of human gonadotrophin (human menopausal gonadotrophin, hMG) is urine donated by members of a religious order in Switzerland. The post-menopausal women living within the convent pool their urine for sale to a company which derives each lot of its product from a batch of the pooled urine. Since the age and endocrine status of each donor to the urine pool changes from batch to batch, each preparation of gonadotrophin is different in chemical composition and in biopotency. Thus there exists a need for a consistent source of human gonadotrophin.
There also exists a need for a source of physiologically correct preparations of human sex steroid hormones. Currently, therapeutic estrogen and progesterone compounds, and analogs thereof, are prepared by standardized chemical synthesis. However, the class of compounds designated "estrogens" produced normally in the human female includes several different formulae and isoforms. Similarly, the class of hormones designated "progestins" includes several different compounds and isoforms. The types and amounts of estrogens and progestins produced naturally vary according to the female's age and overall physiological status, i.e., the specific time point in her menstrual cycle, pregnancy, or menopause. The optimal steroid content for any given therapeutic indication has not been determined. Even if the optimal chemical profile of a sex steroid preparation were determined, chemical synthesis would not be a practical route for production of complex steroid mixtures. Therefore, it is desirable to develop methods which inherently provide a physiologically correct mix of human estrogens and progesterones.
Toxicity testing is another field which scientists have attempted to address through use of in vitro systems (for review see: Nau, H. 1990. in Methods in Developmental Toxicology: Use in Defining Mechanisms and Risk Parameters. Eds. G. L. Kimmel, D. M. Kochhar, CRC Press, pp. 29-43.) To date, in vitro systems based on hormone-secreting cells have been very limited, partly because of the difficulties inherent in maintaining hormone-secreting cells in culture. In theory, the reproductive toxicity of a compound could be assessed by the capacity of the compound to impair hormone-secretion from cells which characteristically secrete a given hormone. A non-human cell line (Chinese hamster ovary, CHO) has been extensively utilized for toxicology analyses, (Tsushimoto, G., et al., 1983 Arch Environ Contam Toxicol 12: 721). Amphibian oocytes have been proposed as a system for the testing of tumor promoting compounds (U.S. Pat. No. 4,983,527; issued Jan. 8, 1991). Xenopus testis explants have been proposed for the testing of mutagenicity and genotoxicity during spermatogenesis (U.S. Pat. No. 4,929,542; issued May 29, 1990). Cell lines established from rat embryo fibroblasts have been proposed as systems for screening for protein inhibitors and activators (U.S. Pat. No. 4,980,281; issued Dec. 25, 1990). Since it is generally recognized that humans have different toxic susceptibilities compared to amphibians and rodents, the above proposed in vitro testing systems are limited by the non-human origins of the cells.
Thus, there exists a need for human hormone-secreting cell lines established in long-term culture for the purposes of 1) production of human hormones, 2) bio-assay of therapeutic gonadotrophins, 3) testing of drug efficacy and design, 4) toxicity testing of drugs and chemicals, and 5) implantation to replace deficient hormone secretion.